Method for purifying biologically active ligate

Abstract

A method for purifying a biologically active ligate. In this method, a ligand bonded to a first phase and having a specific affinity for the ligate is provided. The ligate is provided with a second phase. The first and second phases are then contacted together under conditions in which the ligand and ligate form a complex bonded to the first phase, with the ligand and ligate held together only by one or more non-covalent pressure sensitive bonds. At least a part of the second phase is then separated from the first phase to provide a purified first phase, and the purified first phase subjected to a pressure of at least 300 atmospheres under conditions sufficient to cause release of the ligate from the complex, but not sufficient to cause significant release of the ligand from the first phase. These conditions do not irreversibly cause biological activity of the ligate to be significantly reduced. The ligate released from the complex is then separated from the immediate vicinity of the ligand, and recovered in its biologically active form.

We claim: 
     
       1. A method for purifying a biologically active ligate, comprising the steps of: (a) providing a ligand having a specific affinity for said ligate, said ligand being covalently bonded or adsorbed to a solid support,   (b) providing said ligate within a phase,   (c) contacting said solid support and said phase under conditions in which said ligand and ligate are contacted together to form a complex bonded to said solid support, said ligand and ligate being held together only by one or more non-covalent pressure-sensitive bonds,   (d) separating at least a portion of said phase from said solid support by washing said solid support, to provide a purified solid support comprising said complex bonded to said solid support,   (e) subjecting said purified solid support to a pressure of at least 300 atmospheres under conditions sufficient to cause release of said ligate from said complex, and not sufficient to cause significant release of said ligand from said solid support, said conditions not irreversibly causing the biological activity of said ligate to be significantly reduced,   (f) separating said ligate released from said complex from the immediate vicinity of said ligand, and   (g) recovering said ligate in its biologically active form.   
     
     
       2. The method of claim 1, wherein said subjecting step comprises subjecting said purified solid support to a pressure under conditions insufficient to cause significant reduction in the biological activity of said ligand. 
     
     
       3. The method of claim 1 or 2, wherein said phase is a liquid. 
     
     
       4. The method of claim 1, wherein said subjecting step comprises applying a pressure of at least 500 atmospheres. 
     
     
       5. The method of claim 4, wherein said subjecting step comprises applying a pressure between 500 and 3000 atmospheres. 
     
     
       6. The method of claim 4, wherein said subjecting step comprises applying a pressure between 1000 and 3000 atmospheres. 
     
     
       7. The method of claim 4, wherein said subjecting step comprises applying a pressure of less than 6000 atmospheres. 
     
     
       8. The method of claim 1, wherein said steps of subjecting to a pressure and separating said released ligate are repeated at least once. 
     
     
       9. The method of claim 1, further comprising separating substantially all said phase from said solid support prior to said subjecting step. 
     
     
       10. The method of claim 1, wherein said step of separating said ligate released from said complex is performed while said pressure is maintained. 
     
     
       11. The method of claim 1, wherein said step of separating said ligate released from said complex comprises washing said ligate from said ligand with a solvent. 
     
     
       12. The method of claim 1, wherein said step of separating said ligate released from said complex comprises subjecting said ligate to an electric field to remove said ligate from the vicinity of said ligand.